The broad, long-term objective of this proposal for a cooperative drug discovery group is to meet the critical and urgent need for discovery of more effective and less toxic drugs and regimens for treatment of toxoplasmosis in AIDS patients. Toxoplasmosis has emerged as a major cause of morbidity in patients with AIDS. This often devastating illness in AIDS patients is usually manifested clinically by encephalitis and is frequently associated with untoward neurologic sequelae. Drugs that have proven effective in treatment of toxoplasmosis in AIDS are extremely limited and the two most commonly used are frequently associated with drug reactions sufficiently severe to preclude their further use in a given patient. Thus, there is a real necessity to discover and evaluate new treatment modalities for toxoplasmosis in AIDS patients. The specific aims of the three projects of the proposed collaborative effort are: a) to evaluate newer therapeutic agents for their activity against tachyzoites of T. gondii in vitro and in vivo. Mammalian cells in tissue culture and murine models of toxoplasmic encephalitis will be used in these studies; b) to determine the activity of current and newer therapeutic agents against the tissue cyst form of T. gondii both in vitro and in vivo. None of the presently available drugs has been studied against the cyst form in vitro. For this purpose mice with chronic central nervous system infection will be treated as well as cysts isolated from brains of chronically infected mice; c) to investigate the therapeutic potential of biologic response modifiers alone and in combination with current and newer therapeutic agents for toxoplasmosis. These studies will be performed in vitro in tissue culture as well as in vivo in a model of systemic toxoplasmosis and models of toxoplasmic encephalitis; d) to examine the effect of drugs being used to treat HIV infection on the in vitro and in vivo antitoxoplasma effects of drugs being studied for treatment of toxoplasmosis in AIDS patients; e) to use genetic and molecular techniques to identify the biochemical target(s) of promising new drugs for which the target in T. gondii is not yet known and to determine the molecular changes in these targets which can lead to development of drug resistance. To accomplish these ends, mutants of T. gondii which are resistant to the action of important drugs will be produced and analyzed and the genes identified which give rise to the resistant mutants; f) to study the nature of the glycolytic enzymes of T. gondii and the effect of pyrophosphate analogues and other enzyme inhibitors on viability of the parasite. For this purpose the enzymes of toxoplasma tachyzoites and bradyzoites which are involved in glycolysis will be examined and the PPi-phosphofructokinase characterized.